PROJECT SUMMARY Respiratory diseases are among the leading causes of death worldwide. Respiratory disease caused by smoking, infections, and genetic factors together account for approximately 9.5 million deaths per year. Respiratory infection is the 3rd most common cause of death worldwide, and the leading cause of death in developing countries. Genetic risk factors are significant contributors to lung disease prevalence. For example, cystic fibrosis (CF) is ranked as one of the most widespread life-shortening genetic diseases2-4 with more than 70,000 people currently living with CF. Due to the high incidence; respiratory diseases are also among the most studied medical conditions. The study of respiratory diseases is significantly limited by a lack of suitable in vivo and in vitro models to investigate interactions between the respiratory epithelium, infection, and disease. Unfortunately animal models of lung disease differ significantly from humans in airway development and disease pathology, so often result in inaccurate and significantly flawed models, While some progress has been made in using human cell culture systems for disease modeling and drug discovery, current in vitro models are unable to reproduce the complex spatial morphology and allow biologically relevant cell-cell and cell-matrix interactions. The overall premise for the proposed work is that (a) in vivo animal models often differ significantly from humans in disease pathology and have significant cost limitations; and (b) current in vitro models of respiratory disease and bacterial pathogenesis do not recapitulate the complex tissue components and 3D architecture of the human airway epithelium. This collaborative R01 proposal is motivated by the critical need to address the current gaps and knowledge and overcome the limitations of current in vitro 2D cell culture models for airway disease modeling, therapy development, and pre-clinical testing. Our overall hypothesis is that recapitulation of the in vivo airway microenvironment will provide a more effective in vitro surrogate for airway disease modeling and therapy evaluation. To test this hypothesis, we will first generate bioengineered multicellular 3D airway organoids, supported by a lung extracellular matrix (ECM)-derived biogel with tunable biomechanical properties, to promote multicellular organization and function of healthy airway epithelium (Aim 1). Next we will evaluate whether 3D airway organoids containing CF airway epithelium can model disease pathology (Aim 2). Finally we will use this novel airway disease model to study the pathogenesis of P. aeruginosa (Aim 3). If awarded, this collaborative R01 will allow our multi-disciplinary and multi-institutional team to develop and evaluate 3D airway organoids as a more effective in vitro surrogate for airway disease modeling and therapy evaluation. Likely developments building on this work could include use of patient-specific airway organoid models for testing of personalized disease treatments, combined with collection of clinical data to validate the use of organoids to predict treatment outcomes and guide treatment.